Genetically modified mice expressing humanized PD-1

ABSTRACT

Provided is a method for preparing a PD-1 gene-modified humanized animal model. The method utilizes the CRIPSR/Cas9 technique to replace partial fragments of a mouse PD-1 gene with fragments of a human PD-1 gene using homologous recombination by constructing a targeting vector, thereby preparing a gene-modified humanized mouse. This mouse can normally express a PD-1 protein containing the functional domain of the human PD-1 protein, and can be used as an animal model for mechanism research regarding PD-1, PD-L1 and other signals, for screening regulators, and for toxicological research. The method has an important and high application value in studies on functions of the PD-1 gene and in the development of new drugs.

CLAIM OF PRIORITY

This application is a 371 U.S. National Phase Application of PCTApplication No. PCT/CN2017/090320, filed on Jun. 27, 2017, which claimsthe benefit of Chinese Patent Application 201610487764.7, filed on Jun.28, 2016. The entire contents of the foregoing are incorporated hereinby reference.

FIELD OF THE INVENTION

The present application relates to the field of animal geneticengineering and genetic modification, and in particular, to a method forconstructing a PD-1 gene-modified humanized animal model based on theCRISPR/Cas9 technique and a use thereof in biomedicine.

BACKGROUND OF THE INVENTION

A humanized animal model refers to an animal model that carries humanfunctional genes, cells or tissues. This model is typically used as analternative living model for research on human diseases, and hastremendous advantages and a prospect of extensive applications inunderstanding pathogenesis and drug screening.

To study pathogenesis of complicated human diseases and screen effectivedrugs, a lot of in vivo experiments need to be performed by using idealanimal models. Mice have been one of the mostly used biological models.Considering the differences between mice and humans in physiological,pathological, and many other aspects, however, it is particularlyimportant to construct humanized mouse models that carry humanfunctional genes, cells or tissues. In studies on some human diseases,one method is to use humanized genetic animal models that are preparedby “placing” human genes on chromosomes of rats and mice using a methodof gene modification.

At present, tumor immunotherapy is one of the most promising researchdirections in the field of tumor treatment. The journal Science rankedtumor immunotherapy as No. 1 of Top Ten Science Breakthroughs in 2013.Right now, studies on PD-1/PD-L1 channel inhibitors have attractedparticular attention.

PD-1 (programmed death-1) is mainly expressed on the surface of T cellsand preliminary B cells. Two ligands of PD-1 (PD-L1 and PD-L2) areextensively expressed in antigen-presenting cells (APCs) and others. Theinteraction between PD-1 and its receptors plays an important role innegative regulation of immune response. The expression of PD-L1 proteincan be detected in many human tumor tissues. The micro-environment oftumor sites can induce an expression of PD-L1 on tumor cells. Theexpressed PD-L1 is favorable for genesis and growth of tumors, inducesthe apoptosis of anti-tumor T cells, and then evades the attack from animmune system. By inhibiting the binding of PD-1 and its ligands, tumorcells can be exposed to the attack of an immune system, and then theeffect of killing tumor tissues and treating cancers can be achieved.

Currently, many big domestic and international companies begin tointensify the development of anti-PD-1 drugs. The most known globalpharmaceutical giants are BMS and Merck & Co. Opdivo and Keytruda by thetwo companies have overcome melanoma and lung cancer indications. InNovember 2015, FDA approved the use of Opdivo on advanced renal cellcarcinoma, and the critical Phase III clinical trial of Opdivo on headand neck cancer was successfully completed. The results disclosed onASCO GI 2016 held in January 2016 indicate that these two drugs showpositive therapeutic effect on ductal carcinoma and stomach cancer. Atthe end of 2015, Shanghai Junshi Biosciences Co., Ltd. became the firstcompany in China with a PD-1 monoclonal antibody approved for clinicaluse; in January 2016, BGB-A317, a PD-1 monoclonal antibody by BeiGene(Beijing) Co., Ltd., passed the FDA review for new drug researchapplication and was approved for clinical trials in the U.S.; onFebruary 19, SHR-1210 (a PD-1 monoclonal antibody) for injectiondeveloped by Jiangsu Hengrui Medicine Co., Ltd. was approved formedicine clinical trial with the main indication being solid tumors; onFebruary 22, Walvax Biotechnology issued an announcement that theapplication for clinical research on an anti-PD-1 monoclonal antibodyproduct (genolimzumab injection) developed by its subsidiary GenorBioPharma Co. Ltd. was accepted, and the main potential indicationsthereof included various blood cancers and a variety of solid tumorslike melanoma, non-small lung cancer, and renal cancer. As of March2016, two domestic companies had PD-1 monoclonal antibody drugs approvedfor clinical trials, and two others were in the acceptance process.

The fierce competition among pharmaceutical companies shows the highacceptance of this type of drugs. PD-1 inhibitors could become animportant benchmark in the history of medicine. The US National CancerInstitute (NCI) has listed PD-1 as the second most promising potenttarget in 140 cancer immunotherapy paths and molecules.

Since immunotherapy has relatively significant immunotoxicity, such asdermatitis, colitis, hypophysitis, and the like, the side effect isdirectly related to the degree of immune response and difficult to beavoided through dosage adjustment. A serious adverse reaction,pneumonia, has been reported for nivolumab and MK-3475, both of whichare PD-1 monoclonal antibodies. Therefore, it is very important to havea strict drug screening procedure. Due to the significant differencebetween human physiology and animal physiology, however, experimentresults obtained from animal models sometimes are not applicable tohumans. As humanized animal models can “replicate” some human functionsvery well, this type of models is often used as alternative livingmodels for in vivo research on human diseases. Humanized animal modelshave extensive applications. For example, they are applicable in fieldslike tumors, AIDS, infectious diseases, human degenerative diseases, andblood diseases.

Some PD-1 gene-related animal models have already been developed atpresent. For example, Nihimura et al. prepared BALB/c mice with PD-1knocked out in 2001. These models were mainly used in studies onfunctions of the PD-1 gene and relevant disease mechanisms. Because ofthe tremendous value of the PD-1 gene in applications in fields of tumorand immunotherapy, the present invention is hereby provided in light ofthe insufficiencies and defects of the prior art, so as to make efficacytrials in early stage clinical trials more effective and improve thesuccess rate of research and development.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a PD-1 gene-modifiedhumanized non-human mammal model.

A second object of the present invention is to provide a method forpreparing the PD-1 gene-modified humanized non-human mammal model.

A third object of the present invention is to provide an application ofthe model.

To achieve the objects of the present invention, the following technicalsolution is employed:

1) constructing a targeting vector: obtaining a homology arm from genomeDNA using PCR, and connecting the homology arm with an hPD-1 fragment tobe introduced to the targeting vector;

2) constructing sgRNA: designing and selecting sgRNA according to thetarget site, connecting sgRNA to the vector plasmid, and performing invitro transcription;

3) transferring the targeting vector and the transcribed sgRNA intoembryonic cells, and transplanting the embryonic cells into animals withfalse pregnancy;

4) genotyping the obtained chimeric animals, screening to obtain apositive animal with successful gene knock-in; subsequently, thepositive animal copulating with a wild-type animal to obtain F1generation heterozygotes; and obtaining homozygotes after copulationamong the F1 generation heterozygotes.

Based on the above technical solution, the present invention provides aPD-1 gene-modified humanized animal model, the animal is a non-primatemammal, the genome thereof contains human PD-1 gene fragments and cannormally express PD-1 functional protein, and the expressed proteincontains a functional domain of human PD-1 genes.

The PD-1 gene modification is a gene modification performed on thesecond exon of the PD-1 gene.

Furthermore, the non-primate mammal is a rodent.

Furthermore, the rodent is a mouse.

Preferably, the background of the mouse is C57BL/6.

For the PD-1 gene-modified humanized animal model according to thepresent invention, nucleotides 11053-11385 of its PD-1 gene are replacedby a human DNA fragment, and other mouse PD-1 regions including thetransmembrane region are retained; the human DNA fragment is as shown bySEQ ID NO: 21.

In one example of the present invention, for the provided PD-1gene-modified humanized animal model, nucleotides 11053-11385 of itsPD-1 gene are replaced by a human DNA fragment, and 14/13 bp of the leftand right edges of the second exon and other mouse PD-1 regionsincluding the transmembrane region are retained; the human DNA fragmentis as shown by SEQ ID NO: 21.

The present invention provides the DNA sequence of a humanized mousePD-1 gene, and the protein encoded with the DNA sequence containsfunctional domains of human PD-1 proteins.

The present invention provides the DNA sequence of a humanized mousePD-1 gene, wherein nucleotides 11053-11385 of a mouse PD-1 gene arereplaced by a human DNA fragment, and 14/13 bp of each of the left andright edges of the second exon and other mouse PD-1 regions includingthe transmembrane region are retained; the human DNA fragment is asshown by SEQ ID NO: 21 or is a DNA fragment having 80% or higherhomology with this fragment; the DNA sequence of the humanized mousePD-1 gene contains a nucleotide sequence shown by SEQ ID NO: 14 or a DNAfragment having 80% or higher homology with the DNA sequence of thehumanized mouse PD-1 gene. In one example of the present invention, theupstream and downstream primer sequences used for amplifying the DNAsequence of the humanized mouse PD-1 gene are as shown by SEQ ID NO:22-23.

For the DNA sequence of the humanized mouse PD-1 gene, its CDS sequenceis shown by SEQ ID NO: 15 or a DNA fragment having 80% or higherhomology with SEQ ID NO: 15, its mRNA sequence is shown by SEQ ID NO: 16or a DNA fragment having 80% or higher homology with SEQ ID NO: 16, andthe protein sequence encoded thereby is shown by SEQ ID NO: 17 or aspecific amino acid fragment thereof with unchanged functions. Thepresent invention provides a use of the DNA sequence of a humanizedmouse PD-1 gene in constructing the PD-1 gene-modified humanized animalmodel, wherein nucleotides 11053-11385 of the second exon of the mousePD-1 gene are replaced by a human DNA fragment, and other mouse PD-1regions including the transmembrane region are retained, the human DNAfragment is as shown by SEQ ID NO: 21, and the DNA sequence of thehumanized mouse PD-1 gene contains a nucleotide sequence shown by SEQ IDNO: 14.

Furthermore, in a specific example of the present invention, nucleotides11053-11385 of the second exon of the mouse PD-1 gene are replaced by ahuman DNA fragment, and 14/13 bp is retained for each of the left andright edges.

Furthermore, the use is to inject an in vitro transcription productcontaining an expression vector of the DNA sequence of the humanizedmouse PD-1 gene and Cas9mRNA, and sgRNA plasmid of the second exon ofthe mouse PD-1 gene into a cytoplasm or nucleus of a mouse fertilizedegg, and to transplant the fertilized egg into a receiving female mousefor producing the PD-1 gene-modified humanized mouse model.

The present invention provides sgRNA of the second exon of thespecifically targeted mouse PD-1 gene, and its target site sequence isas shown by SEQ ID NO: 3 or SEQ ID NO: 8.

In the present invention, the upstream and downstream single strandedsequences, as shown by SEQ ID NO: 9-10, respectively, of a target sitesequence as shown by SEQ ID NO: 3 are synthesized; the upstream anddownstream single stranded sequences, as shown by SEQ ID NO: 11-12,respectively, of a target site sequence as shown by SEQ ID NO: 8 aresynthesized.

The CRISPR/Cas9 targeting vector that contains the DNA sequence of thesgRNA falls within the protection scope of the present invention.

The present invention provides a use of the sgRNA or the CRISPR/Cas9targeting vector in preparing a PD-1 gene-modified humanized animalmodel.

In the present invention, a method for preparing a PD-1 gene-modifiedhumanized animal model is provided, the method comprising the followingsteps:

(1) connecting the DNA sequence, 3′ homology arm and 5′ homology arm ofthe humanized mouse PD-1 gene of the present invention to a plasmid, andconstructing a homologous recombinant expression vector of the secondexon of the humanized mouse PD-1 gene; the 5′ homology arm is as shownby SEQ ID NO: 18, and the 3′ homology arm is as shown by SEQ ID NO: 24;

(2) constructing a plasmid of sgRNA of the second exon of the mouse PD-1gene;

(3) injecting the in vitro transcription product from the step (2), theexpression vector from the step (1), and Cas9mRNA into a cytoplasm ornucleus of a mouse fertilized egg, and transplanting the fertilized egginto a receiving female mouse for producing the PD-1 gene-modifiedhumanized mouse model.

In the step (1), the DNA of a wild-type C57BL/6 mouse genome is used asa template for PCR amplification of the 5′ homology arm fragment and the3′ homology arm fragment, and the amplified primer pair sequences are asshown by SEQ ID NO: 19-20 and SEQ ID NO: 25-26, respectively; theplasmid is PV-4G.

The plasmid of sgRNA of the second exon of the mouse PD-1 gene from thestep (2) is obtained by connecting the sgRNA to a pT7-sgRNA plasmid.

The present invention further provides a PD-1 gene-modified humanizedanimal model prepared using the above method.

The present invention provides a use of the animal model, the DNAsequence of the humanized mouse PD-1 gene, the sgRNA for the second exonof the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector in preparingPD-1 or PD-L1 regulators or drugs.

The present invention provides a use of the animal model, the DNAsequence of the humanized mouse PD-1 gene, the sgRNA for the second exonof the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector in researchon PD-1 or PD-L1 signal mechanism.

The present invention provides a use of the animal model, the DNAsequence of the humanized mouse PD-1 gene, the sgRNA for the second exonof the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector inoncological research.

The present invention further relates to sperms, eggs, fertilized eggs,embryos, progenies, tissues or cells from a gene-modified animal,research on PD-1/PD-L1 signal mechanism of the above biologicalmaterial, and use in pre-clinical experiments, such as research on drugtoxicity of PD-1/PD-L1 regulators and screening and development ofPD-1/PD-L1 regulators.

The present invention provides a use of the animal model, the DNAsequence of the humanized mouse PD-1 gene, the sgRNA, or CRISPR/Cas9targeting vector comprising the same in R&D of PD-1 or PD-L1 regulatorsor drugs, in research on PD-1 or PD-L1 signaling mechanism, and inoncological research.

The present invention provides a mouse carrying humanized and modifiedPD-1 genes, wherein its genome contains human PD-1 gene fragments, cannormally express PD-1 functional protein, and the expressed proteincontains a functional domain of human PD-1 genes.

Furthermore, for the mouse carrying humanized and modified PD-1 genes,nucleotides 11053-11385 of the second exon of its PD-1 gene are replacedby a DNA fragment of human PD-1 gene, 14/13 bp is retained for each ofthe left and right edges, and other mouse PD-1 regions including thetransmembrane region are retained; and the human DNA fragment is asshown by SEQ ID NO: 21.

The present invention provides a use of the animal model or the mousecarrying humanized and modified PD-1 genes in preparing a multi-genehumanized animal model.

The multi-gene humanized animal model prepared using the animal model orthe mouse carrying humanized and modified PD-1 genes through copulationor further with a gene editing method also falls within the protectionscope of the present invention.

Furthermore, a use of the multi-gene humanized animal model in drug R&Dor in oncological research falls within the protection scope of thepresent invention. The drugs are monoclonal antibody, bispecificantibody, or polyclonal antibody drugs, as well as other biological orchemical drugs.

The present invention further provides a cell or tissue isolated fromthe animal model or any mouse.

The present invention further provides a use of the animal model, mouse,cell, or tissue in R&D of PD-1 or PD-L1 regulators or drugs, in researchon PD-1 or PD-L1 signaling mechanism, and in oncological research.

The present invention further provides a use of the animal model, mouse,cell, or tissue in evaluating effectiveness of targeted PD-1/PD-L1 drugsor in screening targeted PD-1/PD-L1 drugs.

The present invention further provides a use of the animal model, mouse,cell, or tissue in evaluating effectiveness of joint administration oftargeted PD-1/PD-L1 drugs and other drugs or in screening combined drugsfor joint administration of targeted PD-1/PD-L1 drugs and other drugs.

The present invention further provides a use of the animal model, mouse,cell, or tissue in screening regulators for human PD-1/PD-L1 signalpathway.

The present invention further provides a method for evaluatingeffectiveness of targeted PD-1/PD-L1 drugs, which uses the animal model,mouse, cell, or tissue to evaluate drugs.

The present invention further provides a method for evaluatingeffectiveness of joint administration of drugs targeting PD-1/PD-L1combined with other drugs, which uses the animal model, mouse, cell, ortissue to evaluate combined drugs for joint administration of drugstargeting PD-1/PD-L1 combined with other drugs.

The present invention further provides a method for screening drugstargeting PD-1/PD-L1, which uses the animal model, mouse, cell, ortissue to screen drugs.

The present invention further provides a method for screening jointadministration of drugs targeting PD-1/PD-L1 combined with other drugs,which uses the animal model, mouse, cell, or tissue to screen combineddrugs for joint administration of drugs targeting PD-1/PD-L1 combinedwith other drugs.

Furthermore, the drugs targeting PD-1/PD-L1 are drugs for treatingtumors.

Preferably, the drugs are monoclonal antibody, bispecific antibody, orpolyclonal antibody drugs, as well as other biological or chemicaldrugs. The present invention has the following features: 1) for theanimal model according to the present invention, the second exon of itsPD-1 gene is partially and manually replaced by the second exon of humanPD-1 gene in a manner of DNA sequence homologous recombination; 2) theanimal model according to the present invention can express a PD-1protein containing the functional domain of the human PD-1 protein inthe animal body. The adoption of this method can minimize the impact onhumanization caused by gene splicing and editing; and retain other mousePD-1 gene functional regions including the transmembrane region, so asto avoid abnormality in mouse expression of the PD-1 protein andphysiological functions related to the PD-1 protein as a result of genehumanization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: activity test results for the 5′-terminal targeting site sgRNA(sgRNA1-sgRNA4) and the 3′-terminal targeting site sgRNA(sgRNA5-sgRNA8), wherein Con was a negative control, PC was a positivecontrol, and blank was a blank control.

FIG. 2: a plasmid map of pT7-sgRNA.

FIG. 3: results of enzyme digestion and electrophoresis of TV-PDplasmid, 1 and 2 in the figure representing 2 TV-PD plasmid,respectively.

FIG. 4: PCR identification results of founder mice tails, wherein wt wasa wild-type mouse, nc was a negative control, M was a mark, and 1, 2,and 3 in the figure represent tail PCR identification results of threeF0 mice in Example 9.

FIG. 5: PCR results of F1 generation mice, wherein WT was a wild-typemouse, nc was a negative control, and M was a mark.

FIG. 6: Southern blot results of F1 generation mice, wherein M was amark and WT was wild type.

FIG. 7: flow cytometry results of the PD-1 gene-modified humanizedhybrid mouse.

FIG. 8: flow cytometry results, wherein wild-type C57BL/6 mice andB-hPD-1 homozygous mice were taken, their T-cells in spleens wereactivated through the anti-mouse CD3 antibody, respectively, then theanti-mouse PD-1 antibody mPD-1PE (FIGs. A and B) and the anti-human PD-1antibody hCD47 FITC (FIGs. C and D) were used for cell labeling; it canbe seen through detection and analysis with a flow cytometer that:compared with the control group (FIGs. A and B), cells that express thehuman PD-1 protein can be detected in the spleens of the B-hPD-1homozygous mice; while in the spleens of the C57BL/6 mice, no cells thatexpress the human PD-1 protein were detected.

FIG. 9: RT-PCR detection results, wherein +/+ was a wild-type C57BL/6mouse, H/H was a B-hPD-1 homozygous mouse, and GAPDH was an internalcontrol.

FIG. 10: mouse colon cancer cells MC38 were transplanted into B-hPD-1mice, and the human PD-1 antibody Keytruda was used for anti-tumorefficacy test, and there was no significant difference in average weightincrease between the G1 control group and G2 Keytruda treatment group.

FIG. 11: mouse colon cancer cells MC38 were transplanted into B-hPD-1mice, and the human PD-1 antibody Keytruda was used for anti-tumorefficacy test, the average volume of tumors in experimental animals inthe G2 treatment group was obviously smaller than that of the G1 controlgroup, and the difference was significant.

FIG. 12: mouse colon cancer cells MC38 were transplanted into B-hPD-1mice, and different doses of the human PD-1 antibody Keytruda were usedfor anti-tumor efficacy tests (0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10mg/kg), and there was no significant difference in average weightincrease among experimental animals in the G1-G5 groups.

FIG. 13: mouse colon cancer cells MC38 were transplanted into B-hPD-1mice, and different doses of the human PD-1 antibody Keytruda were usedfor anti-tumor efficacy tests (0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10mg/kg), the average volume of tumors in experimental animals in theG2-G5 groups was obviously smaller than that of the G1 control group,and the difference was significant.

FIG. 14: the modified mouse colon cancer cells MC38-hPDL1 (the PDL1 genehumanized MC38 cells) were transplanted into B-hPD-1 mice, and the humanPD-L1 antibody Tecentriq was used for anti-tumor efficacy test, andthere was no significant difference in average weight increase betweenexperimental animals in the test group and the control group.

FIG. 15: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and the human PD-L1 antibody Tecentriqwas used for anti-tumor efficacy test, the average volume of tumors inexperimental animals in the treatment group was obviously smaller thanthat of the control group, and the difference was significant.

FIG. 16: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and different doses of the human PD-L1antibody Tecentriq were used for anti-tumor efficacy tests (3 mg/kg and10 mg/kg), and there was no significant difference in average weightincrease among experimental animals in the G1-G4 groups.

FIG. 17: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and different doses of the human PD-L1antibody Tecentriq were used for anti-tumor efficacy tests (3 mg/kg and10 mg/kg), the average volume of tumors in experimental animals in theG2-G4 groups was obviously smaller than that of the G1 control group,and the difference was significant.

FIG. 18: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and one or two of Cisplatin, Keytruda,and Tecentriq were used for anti-tumor efficacy test, and there was nosignificant difference in average weight increase among experimentalanimals in the G1-G4 groups.

FIG. 19: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and Cisplatin, Keytruda, and theircombination were used for anti-tumor efficacy tests; the average volumeof tumors in experimental animals in the G2-G6 treatment groups wasobviously smaller than that of the control group, and the volume oftumors in mice in the Cisplatin combined with Keytruda group (G4) wassignificantly smaller than that of mice in the treatment group onlyusing Cisplatin (G2) or the treatment group only using Keytruda (G3),indicating that the therapeutic effect of combination was superior tothe therapeutic effect of separate administration of Cisplatin orKeytruda.

FIG. 20: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, and Cisplatin, Tecentriq, and theircombination were used for anti-tumor efficacy tests; the average volumeof tumors in experimental animals in the treatment groups was obviouslysmaller than that of the control group, and the volume of tumors in micein the Cisplatin combined with Tecentriq group (G6) was significantlysmaller than that of mice in the treatment group only using Cisplatin(G2) or the treatment group only using Tecentriq (G3), indicating thatthe therapeutic effect of combination was superior to the therapeuticeffect of separate administration of Cisplatin or Keytruda.

FIG. 21: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, the positive control Tecentriq or anyone of anti-human PD-L1 antibodies PDL1-Ab1 and PDL1-Ab2 was used foranti-tumor efficacy tests, and there was no significant difference inaverage weight increase among experimental animals in the G1-G4 groups.

FIG. 22: the modified mouse colon cancer cells MC38-hPDL1 weretransplanted into B-hPD-1 mice, the positive control Tecentriq or anyone of anti-human PD-L1 antibodies PDL1-Ab1 and PDL1-Ab2 was used foranti-tumor efficacy tests, wherein there was no significant differencein the volume of tumors between mice in the treatment group using thePDL1-Ab1 antibody (G3) and the treatment group using Tecentriq (G2), andthere was significant difference in the volume of tumors between G2 orG3 and the control group (G1) (P<0.05); on the other hand, the volume oftumors in mice treated with the PDL1-Ab2 antibody (G4) was significantlylarger than that of the Tecentriq treatment group (G2) and the PDL1-Ab1antibody treatment group (G3), indicating that under the same dosage andfrequency, the anti-human PDL1-Ab1 antibody and the positive controlTecentriq have the similar therapeutic effect and have the similareffect on inhibiting tumor growth, while the anti-human PDL1-Ab2antibody has a poorer therapeutic effect to that of Tecentriq or thePDL1-Ab1 antibody.

FIG. 23: the mouse colon cancer cells MC38 were transplanted intoB-hPD-1 mice, five anti-human PD-1 antibodies were used for anti-tumorefficacy tests, and there was no significant difference in averageweight increase among experimental animals in the G1-G6 groups.

FIG. 24: the mouse colon cancer cells MC38 were transplanted intoB-hPD-1 mice, five anti-human PD-1 antibodies were used for anti-tumorefficacy tests, and the volume of tumors in mice in all G2-G6 groupsshrinks to different degrees and/or disappears compared with the controlgroup, indicating that all five anti-human PD-1 monoclonal antibodieshave excellent tumor inhibitory effect.

FIG. 25: flow cytometry results, wherein C57BL/6 mice and doublehumanized CTLA-4/PD-1 heterozygous mice were taken, their T-cells inspleens were activated through the anti-mouse CD3 antibody,respectively, then the anti-mouse CTLA-4 antibody mCTLA-4 APC (FIGs. A,B, C) or the human-source CTLA-4 antibody hCTLA-4 PE (FIGs. D, E, F), orthe anti-mouse PD-1 antibody mPD-1 PE (FIGs. G, H, I) or the anti-humanPD-1 antibody hPD-1 FITC (FIGs. J, K, L), and the anti-mouse T-cellsurface antibody mTcRrβ were used simultaneously for cell labelingT-cell extracellular proteins; cells that express the human CTLA-4 andPD-1 proteins were detected in the spleens of the gene double humanizedCTLA-4/PD-1 heterozygous mice, while no cells that express the humanCTLA-4 or PD-1 protein were detected in the spleens of the C57BL/6control mice.

DETAILED DESCRIPTION OF THE EXAMPLES

Biochemical reagents used in examples of the present application includeEcoRI, EcoRV, HindIII, KpnI, BglII enzymes purchased from NEB with thearticle numbers thereof being R3101M, R3195M, R3104M, R3142M, andR0144M, respectively; NucleoBond® Xtra Maxi Plus EF purchased fromMacherey-Nagel with the article number being 740426; Kanamycin purchasedfrom Amresco with the article number being 0408; Ambion in vitrotranscription kit purchased from Ambion with the article number beingAM1354; AIO and UCA kits purchased from Biocytogen with the articlenumbers being BCG-DX-004 and BCG-DX-001, respectively; Cas9mRNA was fromSIGMA with the article number being CAS9MRNA-1EA; mouse colon cancercells MC38 purchased from EK-Bioscience; E-coli TOP10 competent cellspurchased from Tiangen with the article number being CB104-02;anti-mouse PD-1 from Biolegend with the article number being 135210;Cisplatin from Hospira Australia Pty Ltd.; mouse CD3 antibody from BDwith the article number being 563123; mPD-1 PE from Biolegend with thearticle number being 109104; hPD-1 FITC from Biolegend with the articlenumber being 329904; mTcRrβ PerCP from Biolegend with the article numberbeing 109228; mCTLA-4 APC from Biolegend with the article number being106310; and hCTLA-4 PE from Biolegend with the article number being349906.

Example 1 Design of sgRNAs Targeting PD-1 Gene

The target sequence determines the targeting specificity of small guideRNA (sgRNA) and the efficiency of Cas9 cleavage at the target gene.Therefore, target sequence selection was important for sgRNA vectorconstruction.

Design and synthesize a guide RNA sequence for identifying the5′-terminal targeting site (sgRNA1 to sgRNA4) and the 3′-terminaltargeting site (sgRNA5 to sgRNA8). Using mice as an example, accordingto the function and sequence features of the PD-1 gene, the 5′-terminaltargeting site and the 3′-terminal targeting site were both on thesecond exon of the mouse PD-1 gene, and the target site sequence of eachsgRNA on PD-1 was as follows:

sgRNA-1 target site sequence (SEQ ID NO: 1):5′-agggacctccagggcccattggg-3′sgRNA-2 target site sequence (SEQ ID NO: 2):5′-cagaggtccccaatgggccctgg-3′sgRNA-3 target site sequence (SEQ ID NO: 3):5′-gtagaaggtgagggacctccagg-3′sgRNA-4 target site sequence (SEQ ID NO: 4):5′-ccctcaccttctacccagcctgg-3′sgRNA-5 target site sequence (SEQ ID NO: 5):5′-gcaccccaaggcaaaaatcgagg-3′sgRNA-6 target site sequence (SEQ ID NO: 6):5′-ggagcagagctcgtggtaacagg-3′sgRNA-7 target site sequence (SEQ ID NO: 7):5′-gttaccacgagctctgctccagg-3′sgRNA-8 target site sequence (SEQ ID NO: 8):5′-gcaaaaatcgaggagagccctgg-3′

Example 2 Screening of sgRNAs

The UCA kit was used to detect activities of a plurality of guide sgRNAsscreened from Example 1, and it can be seen from the results that theguide sgRNAs have different activities. See FIG. 1 for the detectionresults.

Two of them, sgRNA3 and sgRNA8, were selected therefrom for subsequentexperiments.

Upstream and downstream single strands of the sgRNAs were synthesized.See Table 1:

TABLE 1 List of sgRNA3 and sgRNA8 sequences sgRNA3 sequence SEQ ID NO: 9Upstream: 5′-tagaaggtgagggacctcc-3′ SEQ ID NO: 10 Downstream:5′-ggaggtccctcaccttcta-3′ sgRNA8 sequence SEQ ID NO: 11 Upstream:5′-caaaaatcgaggagagccc-3′ SEQ ID NO: 12 Downstream:5′-gggctctcctcgatttttg-3′

Example 3 Construction of pT7-sgRNA Plasmid

The source of pT7-sgRNA plasmids: see FIG. 2 for the pT7-sgRNA vectormap. The plasmid backbone was from Takara with the Catalog No. 3299. DNAfragments containing a T7 promoter (taatacgactcactatagg) and sgRNAscaffold were synthesized by a plasmid synthesis company (see SEQ ID NO:13) and were connected to the skeleton vector through enzyme digestion(EcoRI and BamHI). As verified through sequencing by a professionalsequencing company, the results show that the target plasmids wereobtained.

Example 4 Construction of pT7-PD-3 and pT7-PD-8 Vectors

For sgRNA3 and sgRNA8 listed in Table 1, TAGG was added to the 5′ endsof the upstream single strands and AAAC was added to the 5′ ends of thedownstream single strands. After annealing, the upstream and downstreamsingle strands were connected to the pT7-sgRNA plasmids (the plasmid waslinearized first using Bbs1), respectively, to obtain the expressionvectors pT7-PD-3 and pT7-PD-8. See Table 2 for the connection reactionsystem:

TABLE 2 Connection reaction system sgRNA annealing product 1 μL (0.5 μM)pT7-sgRNA vector 1 μL (10 ng) T4 DNA Ligase 1 μL (5 U) 10xT4 DNA Ligasebuffer 1 μL 50% PEG4000 1 μL H₂O Add to 10 μL

The reaction conditions were as follows: connect at room temperature for10 to 30 min, transfer into 30νL it TOP10 competent cells, then take200νL it for coating onto a Kan resistant plate, culture at 37° C. forat least 12 h, select 2 clones for inoculation into an LB medium havingKan resistance (5 ml), and shake to culture at 37° C. and 250 rpm for atleast 12 h.

Randomly select clones for sequencing verification by a sequencingcompany, and select expression vectors pT7-PD-3 and pT7-PD-8 withcorrect connection for subsequent experiments.

Example 5 Sequence Design

Nucleotides 11053-11385 of the second exon of the mouse PD-1 gene (GeneID: 18566) were replaced by a human DNA fragment (SEQ ID NO: 21), andthe ultimately obtained DNA sequence of the modified humanized mousePD-1 (chimeric PD-1 DNA) was shown by SEQ ID NO: 14. SEQ ID NO: 14 onlylists the DNA sequence of the modified portion, wherein the 219-551 bpregion was a human fragment.

According to the above operation, a human DNA fragment was replaced ontothe second exon of the mouse PD-1 gene to ultimately obtain a humanizedmouse. Specifically, the CDS sequence and mRNA sequence of the humanizedmouse PD-1 were shown by SEQ ID NO: 15 and SEQ ID NO: 16, respectively;for SEQ ID NO: 15 (869 bp), the sites 91-423 were a human fragment, andfor SEQ ID NO: 16 (1972 bp), the sites 154-486 were a human fragment;the human/mouse chimeric PD-1 protein sequence was as shown by SEQ IDNO: 17, wherein the 31-141 bp region was a human fragment.

Example 6 Vector Construction

According to the above experimental scheme, upstream primers of 3homologous recombinant fragments, matching downstream primers, andrelevant sequences were designed. Specifically, the DNA of a wild-typeC57BL/6 mouse genome was used as a template for PCR amplification toobtain a 5′ homology arm fragment and a 3′ homology arm fragment; theDNA of a human genome was used as a template for PCR amplification toobtain a human DNA fragment.

The 5′ homology arm (1770 bp): the nucleotide of the site94041502-94043271 (SEQ ID NO:18) with an NCBI login ID being NC000067.6, the upstream primer (SEQ ID NO:19), and the downstream primer(SEQ ID NO:20). The human DNA fragment (333 bp): the nucleotide of thesite 241852634-241852966 (SEQ ID NO:21) with an NCBI login ID being NC000002.12, the upstream primer (SEQ ID NO:22), and the downstream primer(SEQ ID NO:23). The 3′ homology arm (1733 bp): the nucleotide of thesite 94039436-94041168 (SEQ ID NO:24) with an NCBI login ID beingNC_000067.6, the upstream primer (SEQ ID NO:25), and the downstreamprimer (SEQ ID NO:26).

The 5′ homology arm fragment, the 3′ homology arm fragment, and thehuman DNA fragment were connected, via the AlO kit, to the TV-4G plasmidprovided with the kit, to ultimately obtain the vector TV-PD.

Example 7 Vector Verification

Two TV-PD clones in Example 6 were randomly selected, 2 groups ofrestriction endonucleases were used for enzyme digestion verification onthe clones, wherein EcoRI+EcoRV should have 1812 bp+5527 bp, andHindIII+KpnI should have 270 bp+574 bp+1091 bp+5704 bp. See FIG. 3 forthe enzyme digestion results. The enzyme digestion results of plasmids 1and 2 all meet the expectation, indicating that the plasmids havingthese two numbers were verified to be correct by the enzyme digestionverification. Here, No. 2 plasmid was verified to be correct viasequencing by a sequencing company.

Example 8 Microinjection and Embryo Transfer

Fertilized egg of C57BL/6 mice were taken, a microinjector was used toinject the in vitro transcription product of pT7-PD-3 and pT7-PD-8plasmids prepared in Example 4 (using the Ambion in vitro transcriptionkit for transcription according to the instructions), Cas9mRNA and TV-PDplasmids that were premixed into a cytoplasm or nucleus of a mousefertilized egg. Microinjection of embryos was performed according to themethod in the “Mouse Embryo Operation Experiment Manual (3^(rd) Ed),”and the fertilized egg after injection were transferred into a culturemedium for a short period of culture. Then, they were transplanted intothe oviduct of a receiving female mouse to produce gene-modifiedhumanized mice. Founder mice having the C57BL/6 background (i.e., theFounder mice that were the F0 generation) were obtained. The obtainedmice were subjected to hybridization and auto-copulation to increase thepopulation and establish a stable mouse breed. The immune node humanizedmice obtained using this method were named as B-hPD-1.

Example 9 Identification of Gene-Modified Humanized Mice

1. F0 Generation Genotyping

Two pairs of primers were used, respectively, to perform PCR analysis onthe tail genome DNA of the F0 generation B-hPD-1 mice obtained inExample 8. Regarding positions of the primers, L-GT-F was at the leftside of the 5′ homology arm, R-GT-R was at the right side of the 3′homology arm, and R-GT-F and L-GT-R were both on the second exon. The 5′end upstream primer L-GT-F (SEQ ID NO:27), and the downstream primerL-GT-R (SEQ ID NO:28). The 3′ end upstream primer R-GT-F (SEQ ID NO:29),and the downstream primer R-GT-R (SEQ ID NO:30). The PCR reaction system(20 μL) was shown in Table 3:

TABLE 3 10x buffer 2 μL dNTP (2 mM) 2 μL MgSO₄ (25 mM) 0.8 μL Upstreamprimer (10 μM) 0.6 μL Downstream primer (10 μM) 0.6 μL Tail genome DNA200 ng KOD-Plus-(1 U/μL) 0.6 μL

The PCR amplification reaction conditions were shown in Table 4:

TABLE 4 Temperature Time Number of cycles 94° C. 5 min 1 94° C. 30 sec15 67° C. (−0.7° C./cycle) 30 sec 68° C. 1 kb/min 98° C. 10 sec 25 56°C. 30 sec 68° C. 1 kb/min 68° C. 10 min 1  4° C. 10 min 1

If the insertion position of the recombinant vector was correct, thereshould be only one PCR band, the length of the 5′ end primer productshould be 1956 bp, and the length of the 3′ end primer product should be2245 bp. A total of 3 out of the obtained 24 F0 generation mice wereidentified to be positive mice. The PCR identification of the 3 micetails were shown in FIG. 4.

2. F1 Generation Genotyping

The F0 generation mice identified to be positive were subjected tocopulation with wild-type mice to obtain F1 generation mice. PCRanalysis was performed on the tail genome DNA of the F1 generation mice.The PCR system, reaction conditions, and primers re the same as thosefor the F0 generation genotyping. See FIG. 5 for PCR results of the F1generation mice, which indicates that 6 F1 generation mice were positivemice with specific numbers of F1-1, F1-2, F1-3, F1-5, F1-7, and F1-8.

Furthermore, the Southern blot method was used to determine whetherthere was random insertion in the 6 mice determined by PCR to bepositive (F1-1, F1-2, F1-3, F1-5, F1-7, and F1-8). The genomic DNA wasextracted from the mouse tail KpnI and BglII enzymes were selected todigest the genomes, respectively, the digestion products weretransferred to membrane and hybridized. The probes P1 and P2 were on theoutside of the 3′ homology arm and the humanized fragment, respectively.The probe synthesis primers were P1-F (SEQ ID NO:31), P1-R (SEQ IDNO:32); P2-F (SEQ ID NO:33), P2-R (SEQ ID NO:34), respectively.

The genetically engineered mice that have been successfully preparedproduce, via probe hybridization, bands of 2.7 kb or 3.7 kb,respectively, while the wild-type C57BL/6 mouse genome only has bands of7.3 kb or 3.7 kb with no production of hybrid bands.

The experimental results show that the sizes of hybrid bands allconsistent with the expectation, proving that 4 mice were positivehybrid mice with no random insertion and numbers of F1-1, F1-2, F1-3,and F1-5, respectively; it was shown through Southern blot that 2 micewith numbers of F1-7 and F1-8 have random insertion. See FIG. 6 for theSouthern blot test results. This shows that the method according to thepresent invention can construct B-hPD-1 humanized genetically engineeredmice can have stable generations and have no random insertion.

3. Protein Identification

The F1 generation mice obtained in Example 8 were taken, their Tlymphocytes (CD3+) were taken, which were subjected to flow cytometrywith anti-mouse PD-1 and anti-human PD-1 antibodies. From the flowcytometry results (FIG. 7), it can be seen that the percent of thedouble-positive cells obtained by staining the two antibodies anti-humanPD-1 and anti-mouse CD3 was 92%, indicating that the PD-1 gene-modifiedhumanized mouse obtained using this method can express humanized PD-1proteins.

Furthermore, the F1 generation mice obtained in Example 8 were subjectedto mutual copulation to obtain F2 generation PD-1 gene humanizedhomozygotes. One PD-1 gene humanized homozygote (8 week old) wasselected, one wild-type C57BL/6 mouse was taken as a control, 7.5 μgmouse CD3 antibody was administered to the mice through intraperitonealinjection, and after 24 h, the mice were subjected to euthanasia throughneck break. The spleens were collected and grinded. The ground sampleswere then passed through 70 μm cell mesh, the filtered cell suspensionswere centrifuged and the supernatants were discarded; the erythrocytelysis solution was added for lysis of 5 min, and then PBS solution wasadded to neutralize the lysis reaction. The solution was centrifugedagain and the supernatants were discarded. The cells were washed oncewith PBS, then subjected to FACS detection and RT-PCR detection,respectively.

The FACS detection: the anti-mouse PD-1 antibody mPD-1 PE and theanti-mouse T-cell surface antibody mTcRβ, as well as the anti-human PD-1antibody hPD-1 FITC and the anti-mouse T-cell surface antibody mTcRβ,were used simultaneously for staining T-cell extracellular proteins, andafter the cells were cleaned with PBS, flow cytometry was performed todetect protection expression. The flow cytometry results (FIG. 8) showthat compared with the C57BL/6 mice unstimulated and with T-cells inspleens activated through stimulation by the mouse CD3 antibody, cellsthat express the humanized PD-1 protein can be detected in the spleensof the humanized mice for the humanized PD-1 antibody; while in thespleens of the C57BL/6 control mice, no cells that express the humanizedPD-1 protein were detected.

The RT-PCR detection: the total RNA of spleen cells was extracted fromwild-type C57BL/6 mice and B-hPD-1 homozygous mice, and a reversetranscription kit was used for reverse transcription to cDNA.

Primers: mPD-1 RT-PCR F3: (SEQ ID NO:35) and mPD-1 RT-PCR R3: (SEQ IDNO:36) were used to amplify a mouse PD-1 fragment with a size of 297 bp;

Primers hPD-1 RT-PCR F3 (SEQ ID NO:37) and hPD-1 RT-PCR R3 (SEQ IDNO:38) were used to amplify a humanized PD-1 fragment with a size of 297bp.

The PCR reaction system was 20 μL, and the reaction conditions were asfollows: 95° C., 5 min; (95° C., 30 sec; 60° C., 30 sec; 72° C., 30 sec,35 cycles); 72° C., 10 min; keeping temperature constant at 4° C. GAPDHwas used as an internal control.

The experimental results show (FIG. 9) that mRNA expression of the mousePD-1 can be detected in the activated cells of wild-type C57BL/6 mice,and mRNA expression of the humanized PD-1 can be detected in theactivated cells of B-hPD-1 homozygous mice.

Example 10 In Vivo Efficacy Verification on the B-hPD-1 Gene HumanizedAnimal Model

In this Example, 2 drugs for human PD-1/PD-L1 signal channels on themarket that have been extensively verified were selected, which wereKeytruda (pembrolizumab, a humanized monoclonal antibody) from Merck &Co. and Tecentriq (Atezolizumab, a fully humanized monoclonal antibody)from Genentech under Roche.

B-hPD-1 homozygous mice (4-6 weeks old) were taken, mouse colon cancercells MC38 or modified MC38-hPDL1 (i.e., PD-L1 gene humanized MC38cells) were subcutaneously inoculated. When the tumor volume was about100 mm³, the mice were randomly divided into a control group or atreatment group. For the treatment group, one of the above 2 antibodieswas randomly selected and administered at different dosages (0.3-25mg/kg), and the same volume of a blank solvent was injected for thecontrol group. The tumor volume was measured twice a week and the bodyweight were measured for each mouse. Moreover, euthanasia was performedwhen the tumor volume of a single mouse reached 3000 mm³. The specificexperiment scheme was as follows:

Experiment 1 Keytruda antibody efficacy pre-experiment (n=6/group):after subcutaneous inoculation of MC38 cells to mice (5×10⁵/100 μL PBS),the anti-human PD-1 antibody Keytruda was administered to the treatmentgroup at 25 mg/kg through intraperitoneal injection, and the same volumeof a blank solvent was injected for the control group; theadministration frequency was one administration per 3 days for a totalof 6 administrations;

Experiment 2 Keytruda antibody efficacy experiments at differentdosages: after subcutaneous inoculation of MC38 cells to mice (5×10⁵/100μL PBS), the anti-human PD-1 antibody Keytruda was administered to thetreatment group (n=6/group) through intraperitoneal injection atdifferent dosages (0.3-10 mg/kg), and the same volume of a blank solventwas injected for the control group (n=10/group); the administrationfrequency was one administration per 3 days for a total of 6administrations;

Experiment 3 Tecentriq antibody efficacy pre-experiment (n=7/group):subcutaneous inoculation of MC38-PDL1 (5×10⁵/100 μL PBS), the anti-humanPD-1 antibody Tecentriq was administered to the treatment group at 3mg/kg through intraperitoneal injection, and the same volume of a blanksolvent was injected for the control group; the administration frequencywas one administration per week for a total of 2 weeks;

Experiment 4 Tecentriq antibody efficacy experiments at differentdosages (n=5/group): subcutaneous inoculation of MC38-PDL1 (2×10⁵/100 μLPBS), the anti-human PD-1 antibody Tecentriq was administered to thetreatment group through intraperitoneal injection at 1-10 mg/kg, and thesame volume of a blank solvent was injected for the control group; theadministration frequency was one administration per 2 days for a totalof 8 administrations;

Main data and analytical results of all experiments were listed inTables 5, 6, 7, and 8, specifically comprising tumor volumes at the timeof group division and at 10-25 days after group division, tumor volumeswhen the experiments end, situation of mice survival, situation oftumor-free mice, Tumor Growth Inhibition Value (TGI_(TV)), andstatistical difference (P values) in mouse weights and tumor volumesbetween mice in the treatment group and the control group.

Overall, the animals have good health conditions in the experiments ofall groups. At the end of all experiments, all the animals in all groupshave gained weight. There was no significant difference in weightthroughout the entire experiment period between all the treatment groupsand control groups (FIGS. 10, 12, 14, and 16). In terms of the tumormeasurements (FIGS. 11, 13, 15, and 17), however, tumors were growingcontinuously in the experiment period for all mice in the controlgroups. Compared with the control groups thereof, all the treatmentgroups have the volume of tumors shrunk to different degrees and/ordisappears. Therefore, the treatment by either of the two drugs forhuman PD-1/PD-L1 signal channels on the market has significantlyinhibited the growth of tumors in the mice.

Each experiment was specifically evaluated and analyzed. In Experiment 1(see Table 5), all mice in the control group and the treatment groupsurvive at the experiment end-point (Day 26) and had normal weightgains. Compared with the control group, the treatment group does nothave a significant difference in animal weight, indicating that theanimals have good tolerance against Keytruda. Tumors were growingcontinuously in the experiment for all mice in the control group, whileat the experiment end-point, 2 of the 6 mice in the treatment group havetumors disappeared. The average tumor volume of the control group was3168±606 mm³, while the average tumor volume of the treatment group was111±77 mm³. All mice in the treatment group have a tumor volumeobviously smaller than that of the control group, and the difference wassignificant (p<0.05), and TGI_(TV) was 102%. Therefore, it was proventhat according to the administration manner, the anti-human PD-1antibody Keytruda has a significant inhibitory effort on tumors insideB-hPD-1 mice (TGI_(TV)>60%), has good capabilities of treating andinhibiting tumor growth, does not have obvious toxicity on the animals,and has good safety.

TABLE 5 Tumor P values Tumor volume (mm3) Survival free TGI_(TV) TumorExperiment 1 Day 6 Day 18 Day 26 situation situation % Weight volumeControl group 169 ± 16 1450 ± 202 3168 ± 606 100% 0/6 N/A N/A N/ATreatment group 169 ± 24 302 ± 86 111 ± 77 100% 2/6 102 0.05 0.0005

In Experiment 2 (see Table 6), all mice in the control group and thetreatment groups survive at the experiment end-point (Day 32, which wasDay 24 after group division) and had normal weight gains. Compared withthe control group, the treatment groups do not have a significantdifference in animal weight (p>0.05), indicating that the animals havegood tolerance against Keytruda. Similar to Experiment 1, tumors weregrowing continuously in the experiment for all mice in the controlgroup, while at the experiment end-point, 7 of all the 24 mice in thetreatment groups have tumors disappeared. At the experiment end-point,the average tumor volume of the control group was 1589±652 mm³, whilethe average tumor volume of the treatment group was 223±270 mm³, 277±397mm³, 201±186 mm³, and 437±515 mm³ at doses of 10 mg/kg, 3 mg/kg, 1mg/kg, and 0.3 mg/kg, respectively. All mice in the treatment groupshave a tumor volume obviously smaller than that of the control group,and the difference was significant (p<0.05) compared with the tumorvolume of the control group. Overall, the optimal therapeutic effect wasachieved after 3 weeks of administration. After the administration wasstopped, obvious tumor growth occurs in the treatment groups having lowdosage. TGI_(TV) was 92.4%, 89.0%, 94.0%, and 78.2%, respectively,indicating that the anti-human PD-1 antibody Keytruda has a significantinhibitory effort on tumors (TGI_(TV)>60%) and shows differenttherapeutic effects inside B-hPD-1 mice at different dosages.

TABLE 6 Tumor P values Tumor volume (mm³) Survival free TGI_(TV) TumorExperiment 2 Day 8 Day 22 Day 32 situation situation % Weight volumeControl group 100 ± 7  435 ± 210 1589 ± 652  10/10  0/10 N/A N/A N/ATreatment 10 mg/kg 110 ± 28 117 ± 111 223 ± 270 6/6 2/6 92.4 0.1042.6E−04 groups  3 mg/kg 113 ± 25 88 ± 94 277 ± 397 6/6 2/6 89.0 0.3100.001  1 mg/kg 112 ± 27 89 ± 86 201 ± 186 6/6 2/6 94.0 0.678 1.8E−04 0.3mg/kg  113 ± 28 115 ± 116 437 ± 515 6/6 1/6 78.2 0.259 0.002

In Experiment 3 (see Table 7), all mice in the control group and thetreatment group survive at the experiment end-point (Day 20) and hadnormal weight gains. Compared with the control group, the treatmentgroup does not have a significant difference in animal weight (p>0.05),indicating that the animals have good tolerance against Tecentriq.Tumors were growing continuously in the experiment for all mice in thecontrol group, but at this dosage, there was no mouse cured in thetreatment group (i.e., no mice have tumors disappeared). At theexperiment end-point, the average tumor volume of the control group was824±315 mm³, while the average tumor volume of the treatment group was237±89 mm³. All mice in the treatment group have a tumor volumeobviously smaller than that of the control group, and TGI_(TV) was79.7%. Therefore, it was proven that according to the administrationmanner, the anti-human PD-1 antibody Tecentriq has a significantinhibitory effort on tumors inside B-hPD-1 mice (TGI_(TV)>60%), hasstrong capabilities of inhibiting tumor growth, does not have obvioustoxicity on the animals, and has good safety.

TABLE 7 Tumor P values Tumor volume (mm3) Survival free TGI_(TV) TumorExperiment 3 Day 6 Day 17 Day 20 situation situation % Weight volumeControl group 89 ± 6 685 ± 241 824 ± 315 7/7 0/7 N/A N/A N/A Treatmentgroup 88 ± 8 208 ± 72  237 ± 89  7/7 0/7 79.7 0.19 0.098

In Experiment 4 (see Table 8), all mice in the control group and thetreatment groups survive at the experiment end-point (Day 33, which wasDay 24 after group division) and have gained weight. Compared with thecontrol group, the treatment groups do not have a significant differencein animal weight (p>0.05), indicating that the animals have goodtolerance against Tecentriq. Similar to Experiment 3, tumors weregrowing continuously in the experiment for all mice in the controlgroup, while at the experiment end-point, 3 of all the 15 mice in thetreatment groups have tumors disappeared. At the experiment end-point,the average tumor volume of the control group was 2464±1914 mm³, whilethe average tumor volume of the treatment groups was 219±326 mm³,1028±963 mm³, and 1044±432 mm³ at doses of 10 mg/kg, 3 mg/kg, and 1mg/kg, respectively. All mice in the treatment groups have a tumorvolume obviously smaller than that of the control group. TGI_(TV) was97.2%, 93.7%, and 61.2%, respectively, indicating that the anti-humanPD-1 antibody Tecentriq has a significant inhibitory effort on tumors(TGI_(TV)>60%) at different dosages, and higher doses have bettertherapeutic effects. Therefore, it was proven that the anti-human PD-1antibody Tecentriq has different therapeutic effects inside B-hPD-1mice.

TABLE 8 Tumor Tumor volume (mm³) Survival free TGI_(TV) P valuesExperiment 4 Day 8 Day 22 Day 33 situation situation % Day 8 Day 22Control group 147 ± 23 1008 ± 469  2464 ± 1914 5/5 0/5 N/A N/A N/ATreatment 10 mg/kg  153 ± 62 68 ± 75 219 ± 326 5/5 3/5 97.2 0.498 0.032groups 3 mg/kg 147 ± 38 418 ± 268 1028 ± 963  5/5 0/5 93.7 0.907 0.172 1mg/kg 144 ± 31 506 ± 289 1044 ± 432  5/5 0/5 61.2 0.051 0.144

The above research results show that the two human PD-1/PD-L1 antibodiesthat have been extensively used have significant inhibitory and/oreliminating effect on growth of tumors inside B-hPD-1 mice. Therefore,it was proven that the humanized PD-1 animal model can be used toevaluate in vivo the effectiveness of drugs targeting PD-1/PD-L1 and toevaluate therapeutic effects of targeting PD-1/PD-L1.

Example 11 Application of the B-hPD-1 Gene Humanized Animal Model inEfficacy of the Combined Use of Drugs

Clinical research has proven that chemotherapy drugs have significanteffect on a variety of solid tumors in human body, and have featureslike broad anti-tumor spectrum, strong effect, synergistic effect with avariety of anti-tumor drugs with no cross resistance. Jointadministration of monoclonal antibodies for chemotherapy of tumors was amethod that has been extensively used clinically. In this Example, therepresentative human PD-1 antibody Keytruda and the human PD-L1 antibodyTecentriq were administered combined with the first-line chemotherapydrug Cisplatin to prove that the B-hPD-1 mice can be used for researchon the joint administration of the inhibitors for human PD-1/PD-L1signal channels and other drugs.

B-hPD-1 homozygous mice (4-6 weeks old) were taken, 5×10⁵ mouse coloncancer cells MC38-hPDL1 were subcutaneously inoculated at right side(the same as Example 10). When the tumor volume was about 100 mm³, themice were randomly divided into 6 control groups or treatment groups(n=7/group). For the treatment group, one or two of the above 3 drugswere randomly injected, the dosages were 0.1-10 mg/kg, and a blanksolvent was injected for the control group. The tumor volume wasmeasured and the mice were weighed twice a week. Moreover, euthanasiawas performed when the tumor volume of a single mouse reached 3000 mm³.The specific administration or administration combination, dosage,administration manner and frequency were listed in Table 9.

TABLE 9 Group Drug Dose/administiation manner/frequency G1 blank solventintraperitoneal injection: once per 3 days, a total of 6 times G2Cisplatin 10 mg/kg: intravenous push: once per week, a total of 1 weekG3 Keytruda 0.1 mg/kg: intraperitoneal injection: once per 3 days, atotal of 6 times G4 Cisplatin + 10 mg/kg: intravenous push: once perweek, a Keytruda total of 1 week 0.1 mg/kg: intraperitoneal injection:once per 3 days, a total of 6 times G5 Tecentriq 1 mg/kg:intraperitoneal injection: once per 2 days, a total of 8 times G6Cisplatin + 10 mg/kg: intravenous push: once per week, a Tecentriq totalof 1 week 1 mg/kg: intraperitoneal injection: once per 2 days, a totalof 8 times

From the testing results, it can be seen that there was no significantdifference in weight change of mice in the 6 groups (FIG. 18) throughoutthe entire experiment period; from the tumor measurement results (FIGS.19 and 20), however, tumors were growing continuously in the experimentperiod for the mice in the control group (G1). Compared with the controlgroup, the volume of tumors in the 5 treatment groups (G2 to G6)decreased to different degrees, indicating that the tumor growth insidethe mice was significantly inhibited after treatment by different drugsor after joint drug treatment.

Each experiment was specifically evaluated and analyzed. Main data andanalytical results were listed in Table 10, specifically comprisingtumor volumes at the time of group division (8 days after inoculation)and at 10 days after group division, tumor volumes when the experimentsend (25 days after inoculation), situation of mice survival, situationof tumor-free mice, Tumor Growth Inhibition Value (TGI_(TV)), andstatistical difference (P values) in mouse weights and tumor volumesbetween mice in the treatment groups and the control group. Among them,the survival situation was the worst for mice treated with Cisplatinonly (G2), where 2 mice died, followed by the Cisplatin combined withKeytruda group (G4) or the Cisplatin combined with Tecentriq group (G6),each had one mouse died. On the other hand, all mice in the controlgroup (G1) and the treatment groups using antibodies (G3 and G5) surviveat the experiment end-point. At the experiment end-point, the averagetumor volume of the control group (G1) was 1716±789 mm³, the averagetumor volume of the treatment group with Cisplatin only (G2) was 612±510mm³, the average tumor volume of the treatment group with Keytruda only(G3) was 1267±619 mm³, the average tumor volume of the treatment groupof Cisplatin combined with Keytruda (G4) was 496±160 mm³, the averagetumor volume of the treatment group with Tecentriq only (G5) was1234±977 mm³, and the average tumor volume of the treatment group ofCisplatin combined with Tecentriq (G6) was 427±148 mm³. It can be seenthat the tumor volume of the mice in the treatment group withcombination of Cisplatin and Keytruda (G4) was significantly smallerthan that of Cisplatin only (G2) or Keytruda only (G3); the tumor volumeof the mice in the treatment group with combination of Cisplatin andTecentriq (G6) was significantly smaller than that of Cisplatin only(G2) or Tecentriq only (G5). The experimental results show that thechemotherapy drug Cisplatin has certain tumor inhibitory effect, but hascertain toxicity that can lead to mouse death. In addition, the TGI_(TV)value also shows that the therapeutic effect of joint administration ofthe above two monoclonal antibodies and the chemotherapy drug Cisplatinwas superior to the therapeutic effect of separate administration of themonoclonal antibodies or the chemotherapy drug. The simultaneousadministration of the human PD-1 antibody Keytruda or the human PD-L1antibody Tecentriq can more efficiently inhibit the growth of tumorcells.

TABLE 10 Tumor P values Tumor volume (mm³) Survival free TGI_(TV) TumorDay 8 Day 18 Day 25 situation situation % Weight volume Control group G198 ± 23 954 ± 345 1716 ± 789  7/7(100%)  0/7 N/A N/A N/A Treatment G2 98± 24 401 ± 249 612 ± 510 5/7(71.4%) 0/5 68.2 0.062 0.021 groups G3 98 ±30 606 ± 247 1267 ± 619  7/7(100%)  0/7 27.7 0.335 0.260 G4 97 ± 24 311± 117 496 ± 160 6/7(85.7%) 0/6 75.3 0.136 0.004 G5 98 ± 26 688 ± 4091234 ± 977  7/7(100%)  0/7 29.8 0.598 0.330 G6 98 ± 30 242 ± 76  427 ±148 6/7(85.7%) 0/6 79.6 0.067 0.002

The above Examples have proven that the B-hPD-1 mouse model was respondto existing anti-human PD-1/PD-L1 inhibitors and chemotherapy drug orcombination thereof, and has shown dosage correlation with tumor growthinhibition. The following Examples were selected anti-human PD-1/PD-L1inhibitors to further prove that B-hPD-1 mice can be used as alternativeliving model for in vivo research and for screening, evaluating andtreating of human PD-1/PD-L1 pathway regulators.

Example 12 Application of the B-hPD-1 Gene Humanized Animal Model inScreening Anti-Human PD-1/PD-L1 Regulators

Experiment 1: B-hPD-1 homozygous mice (4-6 weeks old) were taken, 5×10⁵mouse colon cancer cells MC38-hPDL1 were subcutaneously inoculated atright side (the same as Example 10). When the tumor volume was about 100mm³, the mice were randomly divided into the control group or treatmentgroups (n=5/group). For the treatment group, the positive controlTecentriq or one of two anti-human PD-L1 antibodies was randomlyselected, all dosages were 3 mg/kg, and a blank solvent was injected forthe control group. The administration manner was intraperitonealinjection, once per 2 days for a total of 8 times. The tumor volume wasmeasured twice a week. Moreover, euthanasia was performed when the tumorvolume of a single mouse reached 3000 mm³.

Main data and analytical results of all experiments were listed in Table11, specifically comprising tumor volumes at the time of group divisionand at 10 days after group division, tumor volumes when the experimentsend, situation of mice survival, situation of tumor-free mice, TumorGrowth Inhibition Value (TGI_(TV)), and statistical difference (Pvalues) in mouse weights and tumor volumes between mice in the treatmentgroups and the control group.

TABLE 11 Tumor P values Tumor volume (mm³) Survival free TGI_(TV) TumorDay 10 Day 20 Day 27 situation situation % Weight volume Control groupG1 111 ± 27 247 ± 80  409 ± 131 5/5 0/5 N/A N/A N/A TreatmentG2(Tecentriq) 111 ± 25 60 ± 70  75 ± 113 5/5 0/5 112.2 0.610 0.003groups G3(PDL1-Ab1) 111 ± 29 89 ± 54 101 ± 128 5/5 2/5 103.3 0.899 0.006G4(PDL1-Ab2) 112 ± 25 180 ± 71  232 ± 88  5/5 0/5 59.8 0.652 0.036

Overall, during the experiments of all groups, the animals were in goodhealth. At each experiment end-point, the animals in all groups hadnormal weight gains. Compared with the control group, none of thetreatment groups has a significant difference in animal weight (P>0.05),indicating that the animals have good tolerance against the threeantibodies. There was no significant difference in weight among the micein all the treatment groups and the control group (FIG. 21) throughoutthe entire experiment period. In terms of the tumor measurement results(FIG. 22), however, tumors were growing continuously in the experimentperiod for all mice in the control group. Compared with the controlgroup, all the treatment groups have the volume of tumors shrunk todifferent degrees and/or disappears, indicating that the two anti-humanPD-L1 monoclonal antibodies have different tumor inhibitory effects, donot have obvious toxicity on the animals, and have good safety.

At the experiment end-point, the average tumor volume of the controlgroup (G1) was 409±131 mm³, the average tumor volume of the Tecentriqtreatment group (G2) was 75±113 mm³, the average tumor volume of thePDL1-Ab1 antibody treatment group (G3) was 101±128 mm³, and the averagetumor volume of the PDL1-Ab2 antibody treatment group (G4) was 232±88mm³. The difference in tumor volume was not significant between mice inthe PDL1-Ab1 antibody treatment group (G3) and the Tecentriq treatmentgroup (G2), while there was a significant difference in tumor volumebetween G2, G3 and the control group (G1) (P<0.05), TGI_(TV) was 112.2%and 103.3%, respectively. On the other hand, the tumor volume of themice in the PDL1-Ab2 antibody treatment group (G4) was significantlybigger than that of the Tecentriq treatment group (G2) and the PDL1-Ab1antibody treatment group (G3), indicating that the anti-human PDL1-Ab1antibody has similar efficacy as that of the positive control Tecentriqunder the same dosage and frequency, leading to equivalent effect oninhibiting tumor growth, while the efficacy of the anti-human PDL1-Ab2antibody was not as good as that of Tecentriq or the PDL1-Ab1 antibody.This experiment proves that the B-hPD-1 mice can be used for screeningdrugs (e.g. antibodies) targeting human PD-L1 and for in vivo efficacydetection.

Experiment 2: B-hPD-1 homozygous mice (4-6 weeks old) were taken, mousecolon cancer cells MC38 (5×10⁵) were subcutaneously inoculated. When thetumor volume was about 100 mm³, the mice were randomly divided into thecontrol group or treatment groups (n=6/group). For the treatment group,one of five anti-human PD-1 antibodies was randomly selected, alldosages were 10 mg/kg, and a blank solvent was injected for the controlgroup. The administration manner was intraperitoneal injection, once per3 days for a total of 6 times. The tumor volume was measured twice aweek. Moreover, euthanasia was performed when the tumor volume of asingle mouse reached 3000 mm³.

Main data and analytical results of all experiments were listed in Table12, specifically comprising tumor volumes before group division andafter group division, tumor volumes when the experiments end, situationof mice survival, situation of tumor-free mice, Tumor Growth InhibitionValue (TGI_(TV)), and statistical difference (P values) in mouse weightsand tumor volumes between mice in the treatment groups and the controlgroup.

TABLE 12 Tumor P values Tumor volume (mm³) Survival free TGI_(TV) TumorDay 6 Day 18 Day 26 situation situation % Weight volume Control group G1169 ± 16 1450 ± 202 3168 ± 606  5/5 0/5 N/A N/A N/A Treatment G2(Ab-A)171 ± 20 330 ± 67 145 ± 58  5/5 2/5 101 0.13 0.0006 groups G3(Ab-B) 168± 18  541 ± 235 744 ± 444 5/5 2/5 81 0.002 0.0091 G4(Ab-C) 168 ± 26  309± 123 301 ± 200 5/5 1/5 95 0.03 0.0012 G5(Ab-D) 167 ± 18 339 ± 69 97 ±45 5/5 3/5 102 0.07 0.0005 G6(Ab-E) 166 ± 18 448 ± 91 493 ± 184 5/5 0/589 0.04 0.0018

Overall, during the experiments of all groups, the animals were in goodhealth. At each experiment end-point, the animals in all groups hadnormal weight gains. Compared with the control group, none of thetreatment groups has a significant difference in animal weight,indicating that the animals have good tolerance against the fiveantibodies. There was no significant difference in weight among the micein all the treatment groups and the control group (FIG. 23) throughoutthe entire experiment period. In terms of the tumor measurement results(FIG. 24), however, tumors were growing continuously in the experimentperiod for all mice in the control group. Compared with the controlgroup, all the treatment groups have the volume of tumors shrunk todifferent degrees and/or disappears, indicating that the five anti-humanPD-1 monoclonal antibodies have different tumor inhibitory effects, donot have obvious toxicity on the animals, and have good safety. Thisexperiment proves that the B-hPD-1 mice can be used for screening drugs(e.g. antibodies) targeting human PD-1 and for in vivo efficacydetection.

Example 13 Preparation and Identification of Double Humanized orMultiple Humanized Mice

The B-hPD-1 mice using this method or already prepared can be furtherused to prepare a double-gene humanized or multi-gene humanized mousemodel. For example, in Example 8 above, the fertilized egg used in themicroinjection and embryo transfer were fertilized egg from other genemodified mice. The fertilized egg of the B-hPD-1 mice can be selectedfor gene editing to further obtain a PD-1 gene humanized and other genemodified double-gene or multi-gene modified or humanized mouse model.Alternatively, the B-hPD-1 homozygous mice or heterozygous mice obtainedusing this method can copulate with other gene modified homozygous orheterozygous mice, the progenies thereof were screened, and according tothe Mendel's genetic law, there was a probability that a PD-1 genehumanized and other gene modified double-gene or multi-gene modified orhumanized mouse model can be obtained. Then, the heterozygous mice weresubjected to mutual copulation to obtain double-gene or multi-genemodified or humanized heterozygous mice.

The preparation of double humanized CTLA-4/PD-1 mice was used as anexample. Since mouse CTLA-4 gene and PD-1 gene were on the samechromosome (#1 chromosome), the fertilized egg of B-hCTLA-4 (CTLA-4 genehumanized) mice were selected for gene editing during the microinjectionin Example 8, and the CTLA-4/PD-1 gene double humanized mice wereultimately obtained through screening positive mouse progenies.

One double humanized CTLA-4/PD-1 heterozygote (6-weeks old) wasselected, two wild-type C57BL/6 mice were selected as the control, 7.5μg mouse CD3 antibody was administered to the mice throughintraperitoneal injection, and after 28 h, the mice were subjected toeuthanasia through neck break. Their spleens were taken, ground andfiltered through a 70 μm cell screen. The filtered cell suspension wascentrifuged, the supernatant was discarded, an erythrocyte lysate wasadded, after 5 min of lysis, and a PBS solution was added to neutralizethe lysis reaction. The solution was centrifuged, the supernatant wasdiscarded, and the cells were washed with PBS once, then the anti-mouseCTLA-4 antibody mCTLA-4 APC (FIGS. 25A, 25B, 25C) or the anti-humanCTLA-4 antibody hCTLA-4 PE (FIGS. 25D, 25E, 25F), or the anti-mouse PD-1antibody mPD-1 PE (FIGS. 25G, 25H, 25I) or the anti-human PD-1 antibodyhPD-1 FITC (FIGS. 25J, 25K, 25L), and the anti-mouse T-cell surfaceantibody mTcRβ were used for staining the isolated T-cell extracellularproteins, and after the cells were washed with PBS, flow cytometry wasperformed to detect protection expression. The flow cytometry resultswere shown in FIG. 25. Compared with the C57BL/6 mice unstimulated andwith T-cells in spleens activated through stimulation by the mouse CD3antibody, T cells that express the human CTLA-4 and PD-1 proteins can bedetected in the spleens of the gene humanized CTLA-4/PD-1 heterozygousmice for both the human-source CTLA-4 antibody and the human-source PD-1antibody, while no T cells that express the human CTLA-4 or PD-1 proteinwere detected in the spleens of the C57BL/6 control mice.

INDUSTRIAL APPLICABILITY

The present invention provides a method for preparing a PD-1gene-modified humanized animal model. The model utilizes the CRIPSR/Cas9technique to replace partial fragments of a mouse PD-1 gene withfragments of a human PD-1 gene in a manner of DNA homologousrecombination by constructing a targeting vector, thereby preparing aPD-1 gene-modified humanized mouse. This mouse can normally express aPD-1 protein containing the functional domain of the human PD-1 protein,and can be used as an animal model for signal mechanism researchregarding PD-1, PD-L1 and other genes and proteins, for screeningindividual or multiple effective regulators and drugs, and forpharmacological research. The method has an important application valuein studies on functions of the PD-1 and PD-L1 genes and in thedevelopment of new drugs.

The invention claimed is:
 1. A genetically modified mouse whose genomecomprises a nucleic acid sequence encoding a chimeric programmed celldeath 1 (PD-1) protein comprising the amino acid sequence of SEQ ID NO:17 operably, linked to a promoter of the mouse PD-1 gene, and whereinthe mouse functionally expresses the chimeric PD-1.
 2. The geneticallymodified mouse of claim 1, wherein the mouse has a C57BL/6 background.3. The genetically modified mouse of claim 1, wherein the genome of themouse comprises a nucleic acid sequence that has at least 80% homologyto the nucleic acid sequence of SEQ ID NO: 21 operably linked to thepromoter of the mouse PD-1 gene.
 4. A cell or tissue isolated from thegenetically modified mouse of claim
 1. 5. The genetically modified mouseof claim 1, wherein the genome of the mouse comprises the nucleic acidsequence of SEQ ID NO: 21 operably linked to the promoter of the mousePD-1 gene.
 6. The genetically modified mouse of claim 1, wherein thegenome of the mouse further comprises a nucleic acid sequence encoding ahumanized CTLA-4.
 7. A method of preparing a genetically modified mouse,the method comprising: 1) providing a plasmid comprising a human PD-1gene fragment that comprises a nucleic acid sequence that has at least80% homology to the nucleic acid sequence of SEQ ID NO: 21 flanked by a5′ homology arm and a 3′ homology arm, wherein the 5′ and 3′ homologyarms target exon 2 of a mouse PD-1 gene; 2) providing two small guideRNAs (sgRNAs) that target the nucleic add sequences of SEQ ID NO: 1-4and SEQ ID NO: 5-8; 3) modifying the genome of a mouse embryo using theplasmid of step 1), the sgRNAs of step 2) and Cas9; and 4) transplantingthe embryo obtained in step 3) into a recipient mouse such a transgenicmouse is obtained, wherein the transgenic mouse has a genome comprisinga nucleic acid sequence encoding a chimeric programmed cell death 1(PD-1) protein comprising the an acid sequence of SEQ ID NO: 17 operablylinked to a promoter of the mouse PD-1 gene, and wherein the mousefunctionally expresses the chimeric PD-1.
 8. The method of claim 7,wherein the 5′ homology arm has the nucleic acid sequence of SEQ ID NO:18, and the 3′ homology arm has the nucleic acid sequence of SEQ ID NO:24.
 9. A method of evaluating a drug for the treatment of cancer, themethod comprising: a) administering a drug to a genetically modifiedmouse that has a tumor, and b) determining whether the drug inhibits thetumor, wherein the genetically modified mouse has a genome comprising anucleic acid sequence encoding a chimeric programmed cell death 1 (PD-1)protein comprising the amino add sequence of SEQ ID NO: 17 operablylinked to a promoter of the mouse PD-1 gene, and wherein the mousefunctionally expresses the chimeric PD-1.
 10. The method of claim 9,wherein the tumor expresses human PD-L1.
 11. The method of claim 9,wherein the genome of the mouse further comprises a nucleic acidsequence encoding a humanized CTLA-4.
 12. The method of claim 9, whereinthe drug is an anti-human PD-1 antibody.
 13. The method of claim 9,wherein the sgRNAs target the nucleic acid sequences of SEQ ID NO: 3 andSEQ ID NO: 8.